Many different types of lateral flow or capillary flow assays and devices are presently on the market. Strip-based tests are, for example, ideal for rapid home and point-of-care testing as well as for detection of various environmental analytes. Lateral flow devices have been widely used as the lateral movement of the test fluid allows its controlled interaction with various reagents which can be located along the test channel allowing control of sequencing and timing of reagent interactions. The most common lateral flow tests available on the market are pregnancy, Chlamydia, Strep throat and HIV tests and tests for routinely screening foods for pathogens (i.e. DuPone™). There are many other applications of lateral flow technology such as those covered in U.S. Pat. Nos. 4,861,711, 4,632,901, 5,656,448, 4,943,522 and U.S. Pat. No. 4,094,647 to name a few.
In lateral flow devices which employ passive means of fluid movement (that is not aided by a fluid pump) fluid movement is induced by capillary forces. These forces originate at the interface between the solid and liquid and can arise in narrow capillaries with smooth surfaces or over a wider cross-sectional area due to the presence of structures which can induce such capillary forces. Typically, such conventional lateral flow assays use lateral flow materials such as nitrocellulose and filter papers which have highly porous structures which induces capillary forces as shown in for example, U.S. Pat. No. 5,601,995. However, such materials have disadvantages in terms of a significant lack of precision largely due to the inherent variability in the production of the nitrocellulose, which leads to further problems in the precise control of movement of liquid samples along the device. Thus, there is a need to provide variations and improvements to such conventional lateral flow assay technology.
For example, many know lateral flow assay devices utilize capillary tubes or channels to direct flow. In US 2002/0187071 and U.S. Pat. No. 5,039,617 (both coagulation assays) for example, the clotting agent is mixed with the sample liquid in a reaction chamber which is separate to the flow path. This is quite a conventional setup and other conventional assays are outlined below. However, it presents various problems where for example the clot can separate from the serum. This separation reduces the effectiveness of the assay to halt or retard fluid flow. As a result, such known capillary tube devices often require long and tortuous flow paths to bring about such retardation of the flow.
Furthermore, these capillary channel-based devices generally have poor surface area to volume ratios which reduce sample contact with the walls of the capillary and exacerbate this problem.
Additionally, the deposition of various reagents on the surface of such capillary tubes is less effective in being dissolved and redistributed by the sample for homogeneous activation of the coagulation response with a resulting inhomogeneity of the clot formation through the sample.
Thus, such know capillary-tube based lateral flow assay devices can present problems during use.
Other problems encountered relate to the materials used for making the lateral flow assay devices. Cyclic polyolefins have been recently used in the manufacture of some lateral flow assay devices. Other materials can be successfully employed in lateral flow test systems including, polystyrene, polyethylene terephthalate (polyester) (PET), polymethylmethacrylate (PMMA), glass, glass fibers, ceramics etc.
Cyclic polyolefins (COP) are thermoplastic resins with an excellent combination of optical and electronic properties. In 1983, while working with norbornene polymer, cyclo-olefin polymers were synthesized from ring-opening polymerization. After hydrogenation of double bonds in the polymer, a glass-clear plastic was created. The high transparency, low specific gravity, low water absorbency, high heat resistance as well as low autofluorescence and high UV transmission of this material make it excellent for biomedical applications (Yamazaki, M. (2004). Industrialization and application development of cyclo-olefin polymer, Journal of Molecular Catalysis a-Chemical, 213 (1): 81; Bhattacharyya, A., Klapperich, C. M. (2006). Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics, Analytical Chemistry, 78 (3): 788). Such materials can be further structured and micropatterned using a host of techniques such as injection moulding and hot embossing to give additional fluid control properties. Advantageously, this type of polymer is chemically inert, which minimizes non-specific biological interactions.
It is known that surface modification of such cyclic polyolefins may result in changing the polymeric material characteristics such as surface tension. Many kinds of surface modification methods aimed at surface wettability enhancement have been described previously. For example, several treatments are aimed at increasing surface tension, by the formation of new functional groups on the polymer surface (Chan, C. M., Ko, T. M., Hiraoka, H. (1996). Polymer surface modification by plasmas and photons, Surface Science Reports, 24 (1-2): 3; Nakao, A., Kaibara, M., Iwaki, M., Suzuki, Y., Kusakabe, M. (1996). Surface characterization of cell adhesion controlled polymer modified by ion bombardment, Applied Surface Science, 101 112). However, in many cases, such treatment methods induce damage to the surface, which reduces its excellent bulk properties. Moreover, the gained benefit of the surface wettability may decrease with time, due to contact with air or water (Suzer, S., Argun, A., Vatansever, O., Aral, O. (1999). XPS and water contact angle measurements on aged and corona-treated PP, Journal of Applied Polymer Science, 74 (7): 1846; Kohler, L., Scaglione, S., Flori, D., Riga, J., Caudano, R. (2001). Ability of a gridless ion source to functionalize polypropylene surfaces by low-energy (60-100 eV) nitrogen ion bombardment. Effects of ageing in air and in water, Nuclear Instruments & Methods in Physics Research Section B-Beam Interactions with Materials and Atoms, 185 267).
In view of these problems, it has been desirable to develop coatings which are water-stable, biocompatible and do not deteriorate over a long period of time. One surface modification technique is UV/ozone treatment. The ozone oxidation can produce peroxides and hydroperoxides with free radicals, and the following immobilization of functional molecules can be performed under thermal or initiator induction. UV exposure in ozone and air was found to impart a highly hydrophilic nature to normally hydrophobic polymeric surfaces (Bhurke, A. S., Askeland, P. A., Drzal, L. T. (2007). Surface modification of polycarbonate by ultraviolet radiation and ozone, Journal of Adhesion, 83 (1-3): 43; Ho, M. H., Lee, J. J., Fan, S. C., Wang, D. M., Hou, L. T., Hsieh, H. J., Lai, J. Y. (2007). Efficient modification on PLLA by ozone treatment for biomedical applications, Macromolecular Bioscience, 7 (4): 467; Suh, H., Hwang, Y. S., Lee, J. E., Han, C. D., Park, J. C. (2001). Behavior of osteoblasts on a type I atelocollagen grafted ozone oxidized poly L-lactic acid membrane, Biomaterials, 22 (3): 219). Flame and corona discharge are other techniques widely used in industry on account of their effectiveness (Martinez-Garcia, A., Sanchez-Reche, A., Gisbert-Soler, S., Cepeda-Jimenez, C. M., Torregrosa-Macia, R., Martin-Martinez, J. M. (2003). Treatment of EVA with corona discharge to improve its adhesion to polychloroprene adhesive, Journal of Adhesion Science and Technology, 17 (1): 47; Strobel, M., Lyons, C. S. (2003). The role of low-molecular-weight oxidized materials in the adhesion properties of corona-treated polypropylene film, Journal of Adhesion Science and Technology, 17 (1): 15). However, none of these techniques are without problems. Above all, they are expensive and the operator is exposed to hazardous conditions. Another well investigated solution to producing functional layers is the attachment of polymer particles by grafting or plasma polymerization (Chen, K. S., Lin, H. R., Chen, S. C., Tsai, J. C., Ku, Y. A. (2006). Long term water adsorption ratio improvement of polypropylene fabric by plasma pre-treatment and graft polymerization, Polymer Journal, 38 (9): 905; Yasuda, H. Plasma Polymerization, Academic Press, New York, 1985, 344) which gives a stable, hydrophilic layer on the plastic surfaces. Graft polymers have been shown to penetrate or partially penetrate the substrate polymer resulting in a thin surface layer (Loh, F. C., Tan, K. L., Kang, E. T., Neoh, K. G., Pun, M. Y. (1995). Near-UV Radiation-Induced Surface Graft Copolymerization of Some O-3-pretreated Conventional Polymer Films, European Polymer Journal, 31 (5): 481; Johansson, B. L., Larsson, A., Ocklind, A., Ohrlund, A. (2002). Characterization of air plasma-treated polymer surfaces by ESCA and contact angle measurements for optimization of surface stability and cell growth, Journal of Applied Polymer Science, 86 (10): 2618). Several types of plasma treatments have been described (Pappas, D., Bujanda, A., Demaree, J. D., Hirvonen, J. K., Kosik, W., Jensen, R., McKnight, S. (2006). Surface modification of polyamide fibers and films using atmospheric plasmas, Surface & Coatings Technology, 201 (7): 4384; Morra, A., Occhiello, E., Garbassi, F. (1990). Wettability and Surface Chemistry of Irradiated PTFE, Angewandte Makromolekulare Chemie, 180 191; Greenwood, O. D., Hopkins, J., Badyal, J. P. S. (1997). Non-isothermal O-2 plasma treatment of phenyl-containing polymers, Macromolecules, 30 (4): 1091. Plasma treatments are known to form polymer radicals on the treated surfaces. However, it has been proven that modification with oxygen plasma alone is not stable over time. This results in the degradation of functional groups created in this process.
For coatings of blood-contacting materials, for example, “silicon-like” films have been widely used. Organic monomer vapours containing silicon alone or in a mixture of other gases, such as O2 are used to create such films (Favia, P., d'Agostino, R. (1998). Plasma treatments and plasma deposition of polymers for biomedical applications, Surface & Coatings Technology, 98 (1-3): 1102). SiOx thin films deposited by PECVD offer several advantages in that they are not only colourless and transparent but are also insoluble, mechanically tough, chemically inert and thermally stable (Leterrier, Y. (2003). Durability of nanosized oxygen-barrier coatings on polymers—Internal stresses, Progress in Materials Science, 48 (1): 1). These characteristics have allowed SiOx films to find expanded application in biomaterials, microelectronics, food and the medical and pharmaceutical industries (Inagaki, N., Tasaka, S., Nakajima, T. (2000). Preparation of oxygen gas barrier polypropylene films by deposition of SiOx films plasma-polymerized from mixture of tetramethoxysilane and oxygen, Journal of Applied Polymer Science, 78 (13): 2389; Bellel, A., Sahli, S., Ziari, Z., Raynaud, P., Segui, Y., Escaich, D. (2006). Wettability of polypropylene films coated with SiOx plasma deposited layers, Surface & Coatings Technology, 201 (1-2): 129; Bieder, A., Gruniger, A., von Rohr, P. R. (2005). Deposition of SiOx diffusion barriers on flexible packaging materials by PECVD, Surface & Coatings Technology, 200 (1-4): 928). For PECVD deposition of SiO2, the simplest and most commonly used mixtures of gases are O2/SiH4, N2O/SiH4. There are several papers reporting good quality SiO2 film deposition in an O2/SiH4 helicon plasma for different applications (Giroultmatlakowski, G., Charles, C., Durandet, A., Boswell, R. W., Armand, S., Persing, H. M., Perry, A. J., Lloyd, RD., Hyde, S. R., Bogsanyi, D. (1994). Deposition of Silicon Dioxide Films Using the Helicon Diffusion Reactor for Integrated Optics Applications, Journal of Vacuum Science & Technology a-Vacuum Surfaces and Films, 12 (5): 2754; Kitayama, D., Nagasawa, H., Kitajima, H., Okamoto, Y., Ikoma, H. (1995). Helicon-Wave-Excited Plasma Treatment of SiOx Films Evaporated on Si Substrate, Japanese Journal of Applied Physics Part 1-Regular Papers Short Notes & Review Papers, 34 (9A): 4747). SiH4 highly diluted in O2 gas feed was widely used in early studies of SiO2 plasma deposition Adams, A. C., Alexander, F. B., Capio, C. D., Smith, T. E. (1981). Characterization of Plasma-Deposited Silicon Dioxide, Journal of the Electrochemical Society, 128 (7): 1545. However, silane is an explosive gas at room temperature and so its use in an industrial environment requires severe safety regulations. Organosilicones, which are relatively inert liquids at room temperature may be preferable to silane (Granier, A., Nicolazo, F., Vallee, C., Goullet, A., Turban, G., Grolleau, B. (1997). Diagnostics in O-2 helicon plasmas for SiO2 deposition, Plasma Sources Science & Technology, 6 (2): 147.). Hexamethyldisiloxane (HMDSO), hexamethyldisilazane (HMDSN), and tetraethoxysilane (TEOS) take a lead as precursors in SiO2 deposition.
Some improvements/modifications to these systems using materials other than nitrocellulose are outlined below. For example, the controlled formation of other microstructures for inducing capillary forces via surface patterning techniques can result in the same capillary force effects, but reduce the inherent variability of materials such as nitrocellulose. For instance, the introduction of periodic structures which protrude from the surface such as micropillars via a range of fabrication techniques (hot embossing, injection moulding, micromachining etc) would induce controlled and reproducible capillary forces. Such defined surface structures can be readily implemented through the fabrication of polymeric plastics such as thermoplastics which are liquid at high temperature and solidify at some lower temperature.
One example of an improved lateral flow assay is a device produced by Amic BV, the 4-cast ‘chip’ as show in FIG. 1. This device is based on a low cost, mass producible, polymer platform fabricated from a range of materials, including cyclic polyolefins, and structured at a micrometer scale using a range of techniques including hot embossing or injection moulding, to form arrays of closely spaced micropillars. This device has been shown to have superior properties over other conventional lateral flow materials utilising, for example, nitrocellulose. Due to the defined, uniform structure of micropillars on the device, fluid flow can be precisely and reproducibly controlled in the device by selecting the size of, and the distance between the micropillars. Thus, fluid is drawn by capillary action through the device. WO 2003/103835, WO 2005/089082 and WO 2006/137785, in the name of AMIC AB, are directed to one such assay method and device. Such devices are used in many different types of assays such as immunoassays, protein microarrays and DNA microarrays. Examples of tests based on Åmic's 4-castchip technology include a fluorescently detected human cardiac Troponin I antigen, total IgG antibody in plasma and c-reactive protein (CRP).
It is known that the controlled movement of fluids is often a prerequisite of a whole host of biological assay devices, where it is often required to move a biological sample to a suitable location at which an assay can be performed, typically by combining the sample with other assay components and/or performing some analysis or measurement at the site. In some instances, the actual movement of the fluid along the assay device is a component of the measurement itself. This is particularly important in the area of blood coagulation monitoring.
The ability of the body to arrest the flow of blood following vascular injury is crucial. The process by which this occurs is termed haemostasis which involves blood coagulation leading to formation of a blood clot or thrombosis. Essentially, a blood clot consists of a plug of platelets in a network of insoluble fibrin particles. Whilst formation of the clot is essential, the persistence of such clots is undesirable and dangerous.
People who suffer from cardiac or vascular diseases and patients that have undergone surgical procedures are at risk of developing blood clots that may result in life-threatening clinical conditions. Such people are often treated with blood-thinning or anticoagulant drugs. However, the amount of anticoagulant in the bloodstream must be maintained at the proper level. Too little may result in unwanted clotting whilst too much can result in haemorrhaging. As a result routine coagulation screening tests have been developed in order to evaluate the coagulation status of blood or plasma. In some instances of blood coagulation monitoring, the measurement of a blood clot is determined by how the blood sample moves in a device during the clotting process. Typically, the clotting cascade is induced by the addition of suitable biochemical reagents and as the blood clots, its resistance to movement is greatly enhanced which can be detected in a range of ways. Some examples of known coagulation assay method and devices are given below.
U.S. Pat. No. 5,372,946 is directed to an apparatus and method for performing a coagulation time test on a sample of blood wherein the blood is deposited in a fluid reservoir and disposable cuvette. In use a blood sample is drawn into a capillary channel by a pumping action and is drawn back and forth through a very narrow aperture. When the clot forms, this movement through the aperture ceases and clotting can be said to have occurred. The testing machine measures the time required each time the blood is caused to traverse the restricted region. However, mechanical assistance in the form of pumping is used in this device and this is undesirable as it adds to the complexity and cost of the device.
U.S. Pat. No. 5,601,995 is directed to an apparatus and method for detecting coagulation in blood samples. In use the test blood sample is allowed to travel through a porous membrane material (nitrocellulose) and the distance it travelled is dependent on the rate of clot formation. This is a standard lateral flow assay conventionally used to monitor coagulation.
U.S. Patent Application Number 2004/0072357, is directed to a device and method for measuring the clotting times in a fluid, typically blood, within a microchannel whereby the onset of clotting is determined by measurement of the rate of change or value of capacitance or impedance between two electrodes situated on either side of the microchannel. In use, the sample is drawn into a polymer microchannel and the distance travelled down the channel before clotting was given as a means of determining the clotting time. However, devices such as US Patent Application No. 2004/0072357, which use open capillary-based systems and employing passive fluid movement suffer from extremely slow filling times which increases the length of time required for the assay. They also have poor surface area to volume characteristics and so have associated problems of mixing reagent with sample in the central lumen of the channel which reduces device efficiency.
Thus, as outlined above one of the major problems with know lateral flow assay devices is to be able to precisely control the movement of liquid samples along such devices. Despite many different solutions proposed there is still a need to improve on conventional lateral flow assay technology, particular in the field of blood coagulation assays. Hence, the present invention is directed to an improved coagulation assay device and method thereof.